The physical and enzymatic properties of DNA replication complexes from Escherichia coli and from HeLa cells are being studied to learn details of the normal and semiconservative mode of DNA replication in vivo. The respective roles of DNA polymerases I, II, and III in normal replication and in excision-repair are being examined in toluenized mutant bacterial strains deficient in one or more of these enzymes. The participation of recombination system enzymes in long-patch repair is being studied. Density and radioactive labeling is used to detect repair synthesis and to distinguish it from semiconservative replication by CsC1 density gradient sedimentation. Our hypothesis that transcription introduces repairable single strand breaks in bacterial DNA is being tested by selective inhibition of transcription of RNA polymerase deficient mutants or by rifampin, puromycin, or chloramphenicol treatment and subsequent examination of the DNA molecular weight distribution by alkaline sucrose gradient sedimentation. Similar studies will be undertaken with HeLa cells and normal human WI38 cells, in culture. The roles of dnaB and recA loci in replication are being examined in isolated preparations of the folded E. coli chromosome. The role of gene 32 protein (unwinding protein) in T4 phage replication is being examined in mutants that produce very low levels of this protein and in sud mutants which partially overcome the presumed deficit.